The present invention is directed to the synthesis and composition of isotopically labeled paclitaxel and related taxane compounds. The present invention is more particularly directed to paclitaxel and related taxane molecules having stable isotopes synthetically incorporated therein at common positions in the chemical structure thereof. The present invention is more specifically directed to a carbon-13 labeled paclitaxel useful as an internal standard for HPLC/MS analysis of biological tissues for quantitation of taxanes. More particularly, the present invention is directed to synthesis and composition of carbon-13 labeled paclitaxel for use as an internal standard for the HPLC/MS analysis of biological tissue samples of clinical and toxicology pharmacokinetic and pharmacodynamic studies.
Various taxane compounds are known to exhibit anti-tumor activity. As a result of this activity, taxanes have received increasing attention in the scientific and medical community. Primary among these is a compound known as xe2x80x9cpaclitaxelxe2x80x9d, which is also referred to in the literature as xe2x80x9ctaxolxe2x80x9d. Paclitaxel has been approved for the chemotherapeutic treatment of several different varieties of tumors, and the clinical trials indicate that paclitaxel promises a broad range of potent anti-leukemic and tumor-inhibiting activity. Paclitaxel has the formula: 
Paclitaxel is a microtubule poison characteristic of a new class of anticancer drugs. In contrast to other natural spindle poisons, such as vinca alkaloids, taxol increases both the assembly and stability of cellular microtubules, and blocks the cell cycle in late G2 and M phases. It has shown significant activity in clinical trials against a number of human tumors, including breast, lung, head, neck, and ovarian carcinomas. Several pharmacokinetic studies, based on high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection, have been reported. In most of these early investigations, the disappearance of taxol from plasma was found to follow a bi-exponential elimination model. In clinical pharmacological studies, several investigators have demonstrated that total urinary excretion of unmetabolized taxol ranges from 1.5% to 9% with a substantial inter-patient variability. These data suggest that renal clearance contributes little to systemic clearance. Conversely, metabolism, biliary excretion and/or extensive tissue binding probably account for the bulk of taxol disposition. Cytochrome P450 enzymes involved in the biotransformation of taxol have been identified using human liver microsomes and various over-expressed human cytochrome P450 isozymes. Preliminary studies showed that 6xe2x88x9d-hydroxytaxol, the major metabolite of taxol in human bile, was also present in plasma. Other putative minor metabolites were also detected in the plasma of human patients. Despite these promising observations, the low concentration of taxol derivatives presents a major difficulty for analysis of paclitaxel and its derivatives and metabolites in biological samples.
Mass spectrometry is a powerful technique for taxoid identification. Electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI), fast atom bombardment (FAB), electrospray (ES) ionization and matrix-assisted laser desorption/ionization (MALDI) techniques have all been used for the mass spectrometric analysis of taxol, taxol-related diterpenoids, and taxol metabolites. Characterization and quantification of taxanes by tandem mass spectrometry (MS/MS) has also been described. All of these mass spectrometry procedures require the separation of the various taxane derivatives from biological fluids. This purification is labor-intensive and requires large volumes of fluids, due to the low concentration of these compounds in human plasma, urine and bile. The direct combination of mass spectrometry with chromatographic techniques offers a new and effective alternative for metabolic studies which involve the analysis of complex biological samples, such as cell culture medium, plasma, bile and urine. HPLC has been successfully interfaced with thermospray (TSP) ionization and atmospheric pressure ionization (API). Recent reports show the application of LC/MS to the analysis of taxanes using thermospray ionization, split-flow LC/ES-MS and LC/ES-MS/MS to screen plant or cell culture extracts.
Pharmacokinetic and pharmacodynamic studies of paclitaxel treatment strategies require extensive analyses of biological samples. Evaluation of oral formulations of paclitaxel for bioavailability following oral dosing in animals requires large numbers of analyses of plasma samples. The average sample preparation plus analysis of HPLC takes 30-60 minutes per sample. Accordingly, it would be desirable to significantly reduce the time for sample preparation and analysis in pharmacokinetic and pharmacodynamic studies of paclitaxel treatment strategies.
A widely used technique of quantitation of analyses by HPLC involves the addition of an internal standard to compensate for various analytical errors. With this approach a known compound at a fixed concentration is added to the unknown sample to give a peak in the chromatograph which can be measured separately. This known compound is used as an internal marker to compensate for the effect of minor variations in separation parameters on peak size, including sample-size fluctuations. However, because the delivery of sample volumes is quite precise with microsampling valves, the main utility of the internal standard technique is in assays that require sample pretreatment (and/or solute derivatization) where variable recoveries of compounds of interest may occur.
To compensate for losses of the compound of interest during sample workup, an internal standard that is structurally similar to the compound(s) of interest is added at a known concentration to the original unknown sample, the pretreatment is carried out, and the resulting sample is analyzed. In this approach any loss of the compound of interest will be accompanied by the loss of an equivalent fraction of internal standard. The accuracy of this approach is obviously dependent on the structural equivalence of the compound(s) of interest and the internal standard i.e., for best results the internal standard and the compound(s) of interest should, among other things, extract equally.
The selection of the internal standard is critical for measurements. An internal standard generally must have a completely resolved peak such that there are no interferences; must elute close to compound(s) of interest (similar kxe2x80x2 values); must behave equivalently to compound(s) of interest for analyses involving pretreatments derivative formation, etc.; must not be present in the original sample; and must be stable such that it is unreactive with sample components, column packing, or the mobile phase. The internal standard must be added at a concentration that will produce a measurement ratio of about unity with compound(s) of interest, and it is desirable for the internal standard to be commercially available in high purity. More than one internal standard may be required for multicomponent mixtures to achieve highest precision.
A practical problem with the internal standard technique is that the standard must be located in a xe2x80x9cvacantxe2x80x9d region in the sample analysis. For simple mixtures this is usually not difficult. However, for complex samples the selection of an internal standard can be tedious. A satisfactory internal standard often is a compound that is structurally related to the compound to be measured (e.g., an isomer or close homolog). The internal standard should have similar kxe2x80x2, solubility, and detection response but be adequately separated from other sample components.
Accordingly, there remains a need to provide a new and improved internal standard for use with pharmacokinetic and pharmacodynamic studies of paclitaxel treatment strategies, and in particular studies utilizing high performance liquid chromatography/mass spectrometry for analysis of paclitaxel in biological samples. There remains a need for an internal standard of paclitaxel for use in high performance liquid chromatography/mass spectrometry which can significantly reduce the time for sample preparation and analysis. The present invention is directed to meeting these needs by providing stable isotopically labeled taxane analogs, and in particular a carbon-13 labeled paclitaxel, and methods for synthesizing the same.
It is an object of the present invention to provide a new and useful internal standard compound for use in the analyses of biological samples for taxane concentrations.
It is a further object of the present invention to provide an isotopically labeled paclitaxel useful as an internal standard in high performance liquid chromatography/mass spectrometry.
It is a further object of the present invention to provide paclitaxel molecules labeled with stable isotopes of carbon and/or hydrogen for use in analyses of paclitaxel treatment strategies.
It is further an object of the present invention to provide a method for producing a new internal standard compound useful as an internal standard in the analyses of biological samples for taxane concentrations.
According to the present invention, then, a chemical compound is provided which comprises an isotopically labeled analog of a standard taxane molecule, such as paclitaxel. The isotopically labeled analog is synthetically formed to have incorporated therein at a selected position a stable isotope of an element that exists at the selected position in the standard taxane molecule. The stable isotope is one which has a mass that is different from the mass of the most abundantly occurring isotope of the element in nature, such that the isotopically labeled analog has a molecular weight different from the molecular weight of the standard taxane molecule.
Exemplary stable isotopes include carbon-13 and deuterium, which may be incorporated at selected positions in the A-ring sidechain of the isotopically labeled analog, such as in an aromatic ring. A plurality of stable isotopes, which may be different isotopes or identical isotopes and particularly carbon-13, may be incorporated in the isotopically labeled analog at corresponding selected positions. The molecular weight of the isotopically labeled analog may differ significantly from the molecular weight of the standard taxane molecule, for example by between about 4 and about 10 amu""s, and preferably about 6 amu""s, such that the isotopically labeled analog is distinguishable by mass spectrometry from the standard taxane molecule.
An isotopically labeled analog according to the present invention may specifically have the formula as follows: 
wherein a majority of carbon atoms in the aromatic ring of the 3xe2x80x2-N-benzoyl group are carbon-13 atoms, and particularly where each carbon atom in the aromatic ring of the 3xe2x80x2-N-benzoyl group may be a carbon-13 atom.
The present invention also provides a method of producing an isotopically labeled taxane molecule. The method comprises the steps of acylating a first compound having the formula as follows: 
wherein
R1 is selected from the group consisting of NH2 and NH3+,
R2 is selected from the group consisting of an acetyl group and a hydroxyl group, and
P1 is a hydroxyl protecting group or hydrogen,
with an acylating agent thereby to form an intermediate compound having a C3xe2x80x2-N-acyl group. The acylating agent is synthetically formed to have incorporated therein at a selected position a stable isotope of an element, wherein the stable isotope has a mass different from the mass of the most abundantly occurring isotope of the element in nature. Where P1 is a hydroxyl protecting group, the intermediate compound is thereafter deprotected by replacing P1 with hydrogen to produce the isotopically labeled taxane molecule. When R2 is a hydroxyl group, the method may include the step of selectively acylating the intermediate compound at the C-10 position prior to the step of deprotecting the intermediate compound.
The step of acylating the first compound to form an intermediate compound having a C3xe2x80x2-N-acyl group may be accomplished by acylating the first compound with benzoyl chloride in the presence of triethylamine, wherein a plurality of carbon atoms in the aromatic ring of the benzoyl group of the benzoyl chloride are carbon-13 atoms. Additionally, the first compound may be formed by coupling an active ester of a protected taxane A-ring sidechain acid with a protected taxane backbone to form a coupled product, and thereafter deprotecting the coupled product to form the first compound. The active ester may have a formula as follows: 
where CBZ is the carbobenzyloxy group, and the protected taxane backbone may be 7-CBZ baccatin III. The step of deprotecting the coupled product may include hydrogenolytic deprotection at the C-7 position to form a 7-deprotected product, followed by hydrogenolytic deprotection at the 3xe2x80x2-N position to form the first compound.
The present invention is also directed to a method of determining the retention of a target taxane in a biological material derived from an organism that has been dosed with the target taxane. The method comprises the steps of taking a selected test sample of the biological material that contains an unknown amount of the target taxane, adding a known amount of an isotopically labeled internal standard to the test sample, processing the test sample to retrieve the target taxane and the internal standard from the test sample, measuring the amount of the target taxane and the amount of the internal standard retrieved from the test sample, and calculating the amount of the target taxane lost during processing of the test sample based on the amount of the target taxane retrieved from the test sample and the ratio of the amount of the internal standard retrieved from the test sample to the known amount of the internal standard added to the test sample.
A plurality of selected test samples of the biological material may be taken. When paclitaxel is the target analyte, the method preferably includes adding an internal standard that is an isotopically labeled analog of paclitaxel, such as one having a plurality of carbon-13 atoms in the aromatic ring of the 3xe2x80x2-N-benzoyl group. The step of processing may include extracting the target taxane and the internal standard from the test sample by partitioning into methyl-t-butyl ether, and may further include separation by liquid chromatography.
The amount of the target taxane and internal standard may be measured using a mass spectrometer operated in positive ion mode. When the target taxane is paclitaxel, the amount of the target taxane is measured by monitoring the transition from 854.4 amu""s to 509.2 amu""s in the mass spectrometer. When the internal standard is an isotopically labeled paclitaxel molecule having a molecular weight about six (6) amu""s greater than the molecular weight of the target paclitaxel, the amount of the internal standard is measured by monitoring the transition from 860.4 amu""s to 509.2 amu""s in the mass spectrometer. The amount of the target taxane lost during processing may be calculated by dividing the amount of the target taxane retrieved from the test sample by the ratio of the amount of the internal standard retrieved from the test sample to the known amount of the internal standard added to the test sample.
These and other objects of the present invention will become more readily appreciated and understood from a consideration of the following detailed description of the exemplary embodiments of the present invention when taken together with the accompanying drawings, in which: